**4.1. Thermopressurized aqueous phosphoric acid pretreatment for partial or total depolymerization of L(h)C biomass**

Thermopressurized aqueous phosphoric (oPA) as pretreatment of L(h)C from different biomasses followed by enzymatic hydrolysis is an efficient approach to further provide free glucose from residual cellulose and immediate free xylose, mannose and the other minor pentose (L-arabinose) and some hexose from the hemicelluloses portion. As phosphoric acid have a higher pKa value than strong acids (pKa: 2.1 against pKa: −3 from H<sup>2</sup> SO4 , pKa: −7 from HCl and pKa: −1.3 from HNO<sup>3</sup> ) it effectively more attractive appeal due to the generation of less carbohydrate dehydration than stronger acids, being possible to carry out the pretreatment of the L(h)C substrate over a wide variety of temperature and pH values. Conversely, the past drawback of phosphoric acid being more expensive than sulfuric acid is now under overcoming considering the aggressive entry of China in the production of several commodities including oPA. A detail, however, is to be taken in account: the moisture content of bulk mineral acids, ranging from 2 to 4, 15, 35 and 63%, respectively for sulfuric, phosphoric, nitric and hydrochloric acids. Nitric acid, due the oxidant action and threat to DNA, has been never considered as a biomass pretreatment. Hydrochloric acid and its toxic fumes offer more serious risk for the labor. Sulfuric acid is a risk for storing, serious burns even by short contact and faster equipment corrosion. So, *inter allia*, we historically from 1980 till nowadays, have been elected phosphoric acid, a gentler proton donor, for all our polysaccharide targets: chronologically from sugar and sorghum bagasses, cassava starch, dahlia tubercles inulin and more recently for microalgae cell wall polysaccharides for the special prospect of nutraceutical oligosaccharides preparation. Except for inulin, the most labile substrate, given its unusual *β*-1,2-furanofructoside linkage (when 10–30 min heating at 70–80°C in open vessels is enough to produce the precious FOS—FructoOligoSacchrides), xylan, mannan and microalgal glucans are pretreated under more severe parameters (thermopressurized reactor) in the range of 2 atm (120°C) to 15.5 (200°C) for a short peaking time (1–2 min) but always keeping the effective oPA concentration with a narrow pH 3.0–1.5 range and more often, at pH 2 or its pKa value (2.1).

Most of the demonstration plants installed worldwide aiming cellulosic ethanol production is based on steam explosion pretreatment, or its variations. One example is POET-DSM Advanced Biofuels that constructed a cellulosic biorefinery alongside the POET Biorefining - Emmetsburg plant. The company contracted ANDRITZ Inc. to supply a two-step biomass treatment process that includes a vertical reactor and a continuous steam explosion (SE) technology to pretreat corn residues (stalks, husks, leaves and cob). In Brazil, GranBio began operations in 2014 with 22 million gallons per year cellulosic ethanol facility, Bioflex 1, using Beta Renewables' PROESA pretreatment process, Novozymes' cellulase enzymes, and DSM's yeasts. In PROESA technology plant, the biomass is pretreated with steam (high temperature and pressure) without chemical addition followed by enzymatic hydrolysis (viscosity reduction and hydrolysis). Some companies have reported difficulties regarding biomass feeding/transport and high degree of equipment wear due to the frictional effect of abrasive materials present in biomass [41]; signaling that this kind of pretreatment still

Other important pretreatments comprise ammonia freeze explosion (AFEX) [42], liquid hot water [43], organosolv and ionic liquids [44], the last one implying in higher costs despite the alternative of catalyst recycling. Also, new pretreatment technologies are constantly being

Thermopressurized aqueous phosphoric (oPA) as pretreatment of L(h)C from different biomasses followed by enzymatic hydrolysis is an efficient approach to further provide free glucose from residual cellulose and immediate free xylose, mannose and the other minor pentose (L-arabinose) and some hexose from the hemicelluloses portion. As phosphoric acid have

less carbohydrate dehydration than stronger acids, being possible to carry out the pretreatment of the L(h)C substrate over a wide variety of temperature and pH values. Conversely, the past drawback of phosphoric acid being more expensive than sulfuric acid is now under overcoming considering the aggressive entry of China in the production of several commodities including oPA. A detail, however, is to be taken in account: the moisture content of bulk mineral acids, ranging from 2 to 4, 15, 35 and 63%, respectively for sulfuric, phosphoric, nitric and hydrochloric acids. Nitric acid, due the oxidant action and threat to DNA, has been never considered as a biomass pretreatment. Hydrochloric acid and its toxic fumes offer more serious risk for the labor. Sulfuric acid is a risk for storing, serious burns even by short contact and faster equipment corrosion. So, *inter allia*, we historically from 1980 till nowadays, have been elected phosphoric acid, a gentler proton donor, for all our polysaccharide targets: chronologically from sugar and sorghum bagasses, cassava starch, dahlia tubercles inulin and more recently for microalgae cell wall polysaccharides for the special prospect of nutraceutical oligosaccharides preparation. Except for inulin, the most labile substrate, given its unusual *β*-1,2-furanofructoside linkage (when 10–30 min heating at 70–80°C in open vessels is

SO4

) it effectively more attractive appeal due to the generation of

, pKa: −7 from

developed, such as sub-/supercritical water [45] and supercritical carbon dioxide [46].

**4.1. Thermopressurized aqueous phosphoric acid pretreatment for partial or total** 

a higher pKa value than strong acids (pKa: 2.1 against pKa: −3 from H<sup>2</sup>

remains as a challenge.

250 Sugarcane - Technology and Research

**depolymerization of L(h)C biomass**

HCl and pKa: −1.3 from HNO<sup>3</sup>

Almost four decades ago, initial studies with L(h)C biomass (rye grass straw) treated with phosphoric acid was carried out to verify the amount of sugar and yeast fermentability after treatments with various concentrations of H2 SO4 , HCl and H3 PO4 aiming to study the feed acceptability by rodents [47]. They have shown that both fermentability (after neutralization with ammonia) and rodent palatability were highest when the straw was treated with a combination of 0.23 N HCl and 0.15 N H3 PO4 (30 min at 121°C), which produced 0.25 g of sugar per g of straw. They also verified that if straw were treated with higher concentrations (>0.5 N) of H2 SO4 or HCl, yeast yield declined probably due to the higher concentration of toxic degradation products of monomeric sugars, such as furfural and HMF.

Prof J.D. Fontana's group at LQBB—Biomasses Chemo/Biotechnological Laboratory formerly at UFPR (Federal University of Paraná) and now at UTFPR (Federal Technological University of Parana, Curitiba-PR, Brazil) has been consolidating for a long time the phosphoric pretreatment technology using very diluted H3 PO4 , alone and under moderated thermopressurization. An initial study focused on the pretreatment of sugarcane and sorghum bagasses with H3 PO4 for the production of bioethanol [48]. The main results from this pretreatment using optimized conditions (0.065% v/v H3 PO4 and 200°C, 3 atm, during 2 min) was a complete or partial hydrolysis of hemicellulose fraction to xylose>xylo-oligosaccharides>arabinose depending on the variation of the severity parameters just before mentioned. Moreover, it was observed improved fermentation of the solubilized pentoses to ethanol and acetic acid by *Pachysolen tannophilus* and to ethanol by *Fusarium oxysporum*. *Pachysolen tannophilus* was the first yeast shown to be capable to convert xylose directly to ethanol under anaerobic conditions (with the concomitant production of xylitol and acetic acid) [49], while *F. oxysporum* is one of the few fungal species reported to ferment plant carbohydrate polymers to ethanol in just one-step process [50]. It was observed that fermentation capability was related to lignin solubilization followed by its removal using ethyl acetate or activated charcoal. Most importantly, phosphoric acid pretreatment on sugarcane and sorghum bagasses allowed almost complete conversion of cellulose to glucose using commercial cellulases produced at that time by Biobras (BIOFERM, Brazil), a Brazilian company that operated a 2G ethanol plant in the end of 1970s and was further acquired by Novo Nordisk's in 2003.

Interestingly, even past >30 years, our oPA technology for sugarcane processing has been used with industrial proposals, as, for example, in the CANEBIOFUEL (Conversion of sugar cane biomass into ethanol) Project, that was funded by the European Commission (FP7-Energy) which planned to obtain a deeper knowledge and a scientific and technological platform for converting sugarcane biomass into fermentable sugars. The project concluded that, in general, lower severity during pretreatment, with lower temperatures and shorter times, result in better glucose yield than the opposite. Primarily based on ease of enzyme hydrolysis it was also found that H3 PO4 is superior to H2 SO4 for the acid catalyzed pretreatment. However, some Brazilian partners in this project applied steam explosion to the pretreatment of sugarcane biomass with almost exactly the same kinetic conditions of our oPA treatment [48]., omitting, unfortunately and consciously, to mention our pioneering publication, as it is completely clear from their below report at Italy.

Current wheat based bioethanol production (first generation) depends significantly on DDGS (distillers dried grains with solubles), a common byproduct that is sold separately as animal feed [57]. Phosphoric acid has been shown as a viable option to maintain substrate quality without contaminating the feed residues with high sulfur levels encountered

wheat straw in laboratory scale and the results validated for the first time in a Biorefinery Demo Plant (BDP), operated by SP (Technical Research Institute of Sweden) at Örnsköldsvik, Sweden [58]. Optimal pretreatment conditions were determined in the laboratory as an acid concentration of 1.75% (w/v) at a temperature of 190°C for 15 min, based on the maximum enzymatic digestibility with the minimum inhibitor release. Enzymatic polysaccharide hydrolysis reached 36% for untreated straw and 86% for straw pretreated with dilute phosphoric acid. Based on this, scale up of the acid phosphoric pretreatment was applied at the biorefinery demonstration plant and an improved efficiency of polysaccharide hydrolysis was obtained (95% of the theoretical maximum). Further sugar fermentation by the Ascomycete *Neurospora intermedia* showed an improvement in the ethanol yield from 29% (with untreated straw) to 94% (with dilute phosphoric acid pretreated straw) of the theoreti-

Starch is the second most abundant polymer in the world [59]. Starch granules are biosynthesized reserve polysaccharide in a broad array of plant tissues and within many plant species. Potatoes and cassava are outstanding starch sources. They are composed of two types of *α*-linked glucans: amylose, a straight chain of *α*-1,4-linked glucopyranosyl units and amylopectin, which has besides *α*-1,4-linked glucopyranosyl units various branch points with *α*-1,6-linkages. A linear polymer of amylose (around 20% of whole starch) can have up to 6000 glucose units, whereas amylopectin (around 80% of the whole starch) is composed of *α*-1,4-linked chains of 10–60 glucose units with *α*-1,6-linked side chains of 15–45 glucose units. Both building blocks represent approximately 98–99% of the starch

Starch may be chemically, enzymatically or physically modified to produce a broth rich in glucose that possess potential use in biotechnological processes, such as fermentation substrate for microorganisms to produce bioethanol, enzymes and other biomolecules. It can be also modified to present novel characteristics, creating innumerous applications, as for example in the food industry, as sweetener or thickening and gelling agent. Enzymatic conversion of starch to free glucose requires the concurrence of two enzymes: *α*-amylase, that yields malto-oligosaccharides and dextrins of varying chain length, and *α*-(1,4)-glucosidase (maltase), which hydrolyses terminal, non-reducing *α*-1,4-linked D-glucose residues with release of free D-glucose. These two enzymes can be replaced by amyloglucosidase (glucoamylase), a single enzyme able to break simultaneously the *α*-D-(1-4) and the *α*-D-(1-6), glycosidic bonds of both poly- and oligosaccharides. Efficient amylase-producing species include those bacteria of genus *Bacillus* (e.g. *B. licheniformis, B. subtilis, B. stearothermophilus, B. amyloliquefaciens*)

**5. Starch hydrolysis with diluted phosphoric acid**

as used. In a recent study, dilute phosphoric acid pretreatment was optimized for

Sugar Versatility—Chemical and Bioprocessing of Many Phytobiomass Polysaccharides Using…

http://dx.doi.org/10.5772/intechopen.75229

253

if H2 SO4

cal maximum.

dry weight [60].

*"Steam explosion of cane bagasse using phosphoric acid catalysis", IBS2010 – 14th Intl. Biotechnology Symposium and Exhibition, Palacongressi, Rimini, Italy; 14–18 Sept, 2010."*

In our another work, aqueous H3 PO4 was used to increase the nutritional value of sugar cane bagasse for cattle feeding [51]. Enhanced ruminal degradability (almost 70%) was obtained by adding 2.9% (w/w) in comparison to 60% achieved with solvolysis with water (197°C,13.5 atm, 4:1 w/w of water). Furthermore, H3 PO4 generates less carbohydrate dehydration and does not have to be washed out prior to fermentation because phosphate can act as an important micronutrient, after partial neutralization with ammonia, for the subsequent fermentation step [51, 52].

Steam treatment of sugarcane bagasse with a low level of phosphoric acid (1% of bagasse dry weight) at elevated temperatures (160–190°C) during 10 min resulted in a total sugar yield ranging from 215 to 299 g/kg bagasse (untreated dry weight) and lower levels of products from sugar degradation (furans and organic acids) in all treatment temperatures (140–190°C) as compared to sulfuric acid [53]. Hemicellulose hydrolysates from treatment temperatures below 180°C could be fermented (slowly) by ethanologenic *E. coli* without the need of purification [53]. This demonstrated low level of potential inhibitors.

In another study, hemicelluloses from sugarcane bagasse were efficiently solubilized (96% and 98% after 8 and 24 min, respectively) using a low concentration of phosphoric acid (0.20%) at 186°C [54]. Enzymatic cellulose conversion of pretreated bagasse using 20 filter paper cellulase units (FPU) g−1 of Novozymes Celluclast® (a commercial cellulase preparation produced by a selected strain of the fungus *Trichoderma reesei*) treated under these conditions of pretreatment produced the highest cellulose conversion of 56.38%. In general low levels of degradation products were achieved; however, minor increase of these products were observed when temperature was elevated to 186°C that can be explained by the high solubilization of hemicellulose fraction at this condition [54] .

Mild phosphoric pretreatment has been also adopted with stream treated substrates. Preimpregnation of *Eucalyptus benthamii* with diluted phosphoric acid followed by steam explosion resulted in an improved selectivity towards hemicellulose hydrolysis (xylose yields of 50–60%), yielding substrates readily susceptible to saccharification with Novozymes Cellic® CTec2 (a commercial enzymatic blend to produce cellulosic ethanol) at relatively high solids (10%) [55].

Results obtained on sugarcane bagasse through a central composite design comparing steam explosion carried out in the absence (autohydrolysis) and presence of phosphoric acid showed that phosphoric acid catalysis (19 mg g−1) resulted in better glucan yields under milder conditions (180°C, 5 min) [56]. Phosphoric acid catalysis produced steam-treated substrates with good susceptibility to enzymatic hydrolysis (30 mg g−1 Cellic® CTec2, at 8% of substrate consistency) yielding in average 75% of glucose.

Current wheat based bioethanol production (first generation) depends significantly on DDGS (distillers dried grains with solubles), a common byproduct that is sold separately as animal feed [57]. Phosphoric acid has been shown as a viable option to maintain substrate quality without contaminating the feed residues with high sulfur levels encountered if H2 SO4 as used. In a recent study, dilute phosphoric acid pretreatment was optimized for wheat straw in laboratory scale and the results validated for the first time in a Biorefinery Demo Plant (BDP), operated by SP (Technical Research Institute of Sweden) at Örnsköldsvik, Sweden [58]. Optimal pretreatment conditions were determined in the laboratory as an acid concentration of 1.75% (w/v) at a temperature of 190°C for 15 min, based on the maximum enzymatic digestibility with the minimum inhibitor release. Enzymatic polysaccharide hydrolysis reached 36% for untreated straw and 86% for straw pretreated with dilute phosphoric acid. Based on this, scale up of the acid phosphoric pretreatment was applied at the biorefinery demonstration plant and an improved efficiency of polysaccharide hydrolysis was obtained (95% of the theoretical maximum). Further sugar fermentation by the Ascomycete *Neurospora intermedia* showed an improvement in the ethanol yield from 29% (with untreated straw) to 94% (with dilute phosphoric acid pretreated straw) of the theoretical maximum.
